Diese Arbeit wurde durch ein Marie Curie Reintegration Grant (IRG249084) unterstützt. Wir danken allen Mitgliedern der Kurant Labor.

Fresszellen und apoptotischen Zellen: Um die Dynamik des apoptotischen Clearance in vivo zwei Populationen von Zellen markiert werden folgen müssen. Makrophagen, Glia und Ektoderm 8: phagocytic Zellpopulationen verwenden wir eine Drosophila Zeile mit der Simu-cytGFP Marker, die ausschließlich Fresszellen Etiketten im Embryo zu markieren. Man kann verschiedene Marker für Fresszellen, einschließlich Linien, die eine hemocyte-spezifische Gal4 Treiber (crq-Gal4) oder Glia-spezifische Gal4 Treiber (Repo-Gal4) und einem genetisch kodierten fluoreszierenden Reporter unter UAS-Kontrolle (UAS-GFP) verwenden. Um apoptotischen Zellen während apoptotischen Clearance Monitor verwenden wir verschiedene Apoptose / Phagozytose Marker. Annexin V (Molecular Probes) dient als ein früher Marker für apoptotische Zellen 10; Phiphilux (OncoImmunin) eine fluorogene Caspase-3-Substrat, die als später Apoptose verwendet wirdsis Marker und LysoTracker (Molecular Probes) arbeitet als phagosome Marker. Wir injizieren diese Reagenzien mit dem Mikroinjektionssystem PicoPump PV 820. 1. Getting Ready <ol><li> Heptane Kleber: Rollen Sie doppelseitiges Klebeband und legen Sie so viel Sie können in einem Szintillationsfläschchen, mit Heptan füllen, verschließen Sie das Fläschchen mit Parafilm und Rock für 24 Stunden. In Heptan, wenn der Kleber ist zu dick. </li><li> Wir lassen die Fliegen auf einer vorgewärmten Traubensaft Agarplatte + Hefe für 2 Std. fügen lag, und übertragen Sie dann die Platte auf 25 ° C für 10-12 Stunden (abhängig von der gewünschten Stufe) vor der Mikroinjektion und Zeitrafferaufnahmen der injizierten Embryonen. Wir sammeln Embryonen von den Agar-Platten bei 25 ° C gehalten, wodurch Embryonen aus der Stufe 15 bzw. 16 der Entwicklung. </li><li> Needle Zubereitung: Um Nadeln zur Injektion, verwenden wir eine &quot;Sutter Instrument &#39;Nadel Abzieher und dünnwandigen Glasfilamenten (FHC Kapillar-Rohr). Die Spitze der Kapillare gebündelt ist pleiten zu einem Durchmesser von 0,5-2 um. </li><li> Deckglas mit Leim: Mit einer Pipettenspitze dispergieren einen Tropfen der Heptan Klebstoff in einer Linie in der Mitte des Deckglases. Lassen Sie es für ein paar Sekunden trocknen. Bereiten Sie ein paar von diesen Deckgläser. </li></ol> 2. Embryo Vorbereitung <ol><li> Sammeln Embryonen aus Agar-Platten und übertragen sie auf eine Zelle Sieb (SPL Life Sciences) mit einem sauberen Pinsel und fließendes Wasser. Das Sieb wird über ein Becherglas gehalten, um Abwasser zu sammeln. </li><li> Waschen Embryonen in der Zelle Sieb mit Wasser, bis alle die Hefe Paste entfernt wird. </li><li> Trocknen Sie das überschüssige Wasser durch Wischen Sie die Außenseite des Filters mit Kimwipes. </li><li> Legen Sie die Zellsieb in einem sauberen Petrischale und fügen Sie genug 50% Bleichmittel auf Embryonen in der Zelle Sieb abdecken. Dechorionate die Embryonen für 2 min gelegentlich (2-3 mal) Rühren sie. </li><li> Ausgiebig mit Wasser spülen, bis Embryonen lose das Bleichmittel Geruch. </li><li> Legen Sie die Zelle Sieb in eine sauberePetrischale und decken die Embryonen mit Wasser zu verhindern Austrocknen. </li><li> Bevor Embryonen Einstellung, laden 1 ul der gewünschten Reagenz in zwei Nadeln (manchmal eine Nadel kann verstopft sein). Es dauert ca. 5 Minuten für die Flüssigkeit, um die Spitze der Nadel zu erreichen. </li><li> Schneiden Sie ein rechteckiges Stück Traubensaft Agar und legen Sie sie auf einem Objektträger. Übertragen dechorionated Embryonen aus der Petrischale auf dem Agar Stück mit einem sauberen Pinsel. </li><li> Unter einem fluoreszierenden Dissektionsmikroskop richtig wählen inszeniert Embryonen Expression des GFP-Marker mit einem nassen Pinsel und legen jedem Embryo nahe an den Rand des Agar Stück in einer Reihe nacheinander (Abbildung 1A). Die Embryonen sollten mit ihrer Bauchseite nach oben eingelegt werden zur Bildgebung Phagozytose durch Gliazellen im zentralen Nervensystem (ZNS). </li><li> Richten Sie etwa 10 Embryonen in einer Zeile. </li><li> Befestigen der Embryonen auf einem Deckglas in der Mitte mit einem Streifen der Klebstoff Heptan (1B, C beschichtet </strong&gt;). </li><li> Legen Sie das Deckglas in der Dehydratisierung Kammer für ca. 5 min (dies hängt von der Luftfeuchtigkeit des Raumes und der Menge, die injiziert werden). </li><li> Nach dem Trocknen vertuschen die Embryonen mit Halocarbonöl 700 (Sigma) zur Vermeidung weiterer Austrocknung. </li><li> Zeigen das Deckglas auf einem Objektträger mit den Embryonen nach oben. Sie können einen Tropfen Wasser auf der Folie zu verhindern Bewegung des Deckglas gelegt. Embryos sind nun bereit für die Injektion. Injektionsstelle ist typischerweise auf der lateralen Seite in der Mitte des Embryos. </li></ol> 3. Embryo Injection <ol><li> Bringen Sie die Nadel in einem Mikromanipulators. </li><li> Um brechen die Nadelspitze setzen den Rand des Deckglases in das Blickfeld und stellen Sie die Nadel auf die gleiche Bildebene. Sehr vorsichtig bewegen das Deckglas, bis er die Nadelspitze trifft und bricht sie. </li><li> Die Konzentration auf die Embryonen, legen Sie die Nadel in die Öl-und sicherzustellen, dass Sie eine flüssige Tropfen aus der Nadel. Es bedeutet, dass die Nadelspitze hatgebrochen gut. </li><li> Ohne Berührung der Nadelhalter bewegen den Embryo in die Nadel und injizieren einen Tropfen in den Embryo. Bewegen Sie den Embryo entfernt und fahren mit dem nächsten, bis alle Embryonen injiziert werden. </li></ol> 4. Imaging Wir Bild die Embryonen auf einem invertierten konfokalen Mikroskop mit einem 40x oder 100x Objektiv. Wir suchen nach einem schön positioniert Embryo mit dem ZNS in der Mitte, die eine starke GFP-Expression und eine gute Kennzeichnung von apoptotischen Zellen nach der Injektion zeigt. Für Zeitrafferaufnahmen von lebenden Embryonen wählen wir normalerweise 5 oder 6 konfokalen Scheiben (2 um dicke jeweils). Danach machen wir einen Vorsprung von 3 Scheiben (6 um Dicke), um ganze Zellen zu beobachten. Wir verwenden Marker für apoptotische Zellen, die stabil sind und nicht bleichen leicht. Um GFP Bleichen machen wir Aufnahmen in Intervallen von 60 sec zu vermeiden. In einigen Fällen Embryonen können rollenwährend der Zeit der Videoaufnahme. Daher werfen wir einen Blick einmal in eine Weile bei der Aufnahme, um zu stoppen und neu zu starten, wenn nötig.

<ol>
	<li>Kinchen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+to+die+for:+signaling+to+apoptotic+cell+removal+in+worm,+fly+and+mouse.">A model to die for: signaling to apoptotic cell removal in worm, fly and mouse.</a> Apoptosis. 15, 998-1006 (2010).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+maturation:+going+through+the+acid+test.">Phagosome maturation: going through the acid test.</a> Nat. Rev. Mol. Cell Biol. 9, 781-795 (2008).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+signaling:+you+can+touch,+but+you+can't+eat.">Phagocytic signaling: you can touch, but you can't eat.</a> Curr. Biol. 18, 521-524 (2008).</li><li>Stuart, L. M., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis:+elegant+complexity.">Phagocytosis: elegant complexity.</a> Immunity. 22, 539-550 (2005).</li><li>Lauber, K., Blumenthal, S. G., Waibel, M., Wesselborg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+cells:+getting+rid+of+the+corpses.">Clearance of apoptotic cells: getting rid of the corpses.</a> Mol. Cell. 14, 277-287 (2004).</li><li>Henson, P. M., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cell+removal+in+development+and+tissue+homeostasis.">Apoptotic cell removal in development and tissue homeostasis.</a> Trends Immunol. 27, 244-250 (2006).</li><li>Kurant, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+the+CNS+clear:+glial+phagocytic+functions+in+Drosophila.">Keeping the CNS clear: glial phagocytic functions in Drosophila.</a> Glia. 59, 1304-1311 (2011).</li><li>Kurant, E., Axelrod, S., Leaman, D., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six-microns-under+acts+upstream+of+Draper+in+the+glial+phagocytosis+of+apoptotic+neurons.">Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons.</a> Cell. 133, 498-509 (2008).</li><li>Mergliano, J., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-independent+cell+engulfment+mirrors+cell+death+pattern+in+Drosophila+embryos.">Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos.</a> Development. 130, 5779-5789 (2003).</li><li>van den Eijnde, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+exposure+of+phosphatidylserine+during+apoptosis+is+phylogenetically+conserved.">Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved.</a> Apoptosis. 3, 9-16 (1998).</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Apoptotische Clearance ist ein kritischer letzten Stadium der Apoptose, die sehr dynamisch ist. Daher Echtzeit Studien des Verfahrens sind von größter Bedeutung. Hier beschreiben wir ein Protokoll, das die Überwachung des apoptotischen Clearance ermöglicht in lebenden Drosophila Embryonen entwickeln. In diesem Verfahren sind zwei Populationen von Zellen mit spezifischen Markern markiert werden: apoptotischen Zellen und Phagozyten. Die Simulation cytGFP Reporter eignet sich zur Überwachung phagocyt...

Die ordnungsgemäße Beseitigung von unerwünschten oder anomalen Zellen durch Apoptose und anschließende Phagozytose ist von entscheidender Bedeutung für die embryonale Entwicklung sowie Gewebshomöostase beim Erwachsenen. Phagozytose von apoptotischen Zellen Apoptose oder Clearance ist ein sehr dynamischer Prozess, der in vier Stufen verläuft: (1) Rekrutierung von Phagozyten der apoptotischen Zelle (&quot;Find-me&quot;), (2) die Anerkennung der Zelle als Ziel für die Phagozytose ( &quot;eat-me &#39;) und (3) engulfment, gefolgt von (4) Phagosomenreifung und Abbau der apoptotischen Teilchen 1-5. Es gibt zwei Arten von Fresszellen: &quot;professionelle&quot; Makrophagen und unreifen dendritischen Zellen, und die &quot;nicht-professionellen&quot; Gewebe-resident benachbarten Zellen, die entscheidend für apoptotischen Clearance bei Metazoen Entwicklung 6,7 sind. In Drosophila Entwicklung erfolgt die Eliminierung von überflüssigen Zellen durch Apoptose in drei main Phasen, zuerst in der Mitte bis Ende der Embryo, dann in der Mitte der Puppe, und wieder im frühen Erwachsenenalter. Während der Embryogenese apoptotischer Partikel werden durch &quot;professionelle&quot; Phagozyten, Makrophagen und durch die &quot;nicht-professionellen&quot; Ektoderm und Glia 8,9 entfernt. In unserer bisherigen Arbeit haben wir einen phagocytic Rezeptor, Six-Mikron-under (SIMU), die ausschließlich in Fresszellen ausgedrückt während der Embryogenese identifiziert. Mit der Simulations-Promotor erzielten wir ein spezifischer Marker für phagocytic Zellpopulationen (Simu-cytGFP), die uns zu Makrophagen, Ektoderm und Glia gleichzeitig überwachen in einer Live-entwickelnden Embryo 8 ermöglicht. Zusätzlich kann durch Verwendung spezifischer Marker für apoptotische Zellen in verschiedenen Stadien der Apoptose, sind wir in der Lage, um die Dynamik des apoptotischen Clearance in vivo zu folgen.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Annexin V </td>
 <td>Molecular Probes</td>
 <td>A35108</td>
 <td></td>
 </tr>
 <tr>
 <td>PhiPhiLux G2D2</td>
 <td>OncoImmunin</td>
 <td>A304R2G-5</td>
 <td></td>
 </tr>
 <tr>
 <td>LysoTracker</td>
 <td>Molecular Probes</td>
 <td>L-7528</td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon oil 700</td>
 <td>Sigma</td>
 <td>H8898</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>Capillary tubing</td>
 <td>FHC </td>
 <td>30-30-0</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Strainer</td>
 <td>SPL</td>
 <td>93100</td>
 <td></td>
 </tr>
 <tr>
 <td>Paintbrush</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

live imaging of apoptotic cell clearance during drosophila embryogenesis

Die ordnungsgemäße Beseitigung von unerwünschten oder anomalen Zellen durch Apoptose und anschließende Phagozytose (apoptotischen Clearance) ist entscheidend für die normale Entwicklung in allen vielzelligen Organismen. Apoptotische Clearance ist ein sehr dynamischer Prozess eng mit Zelltod assoziiert; unengulfed apoptotischen Zellen sind kaum in vivo unter normalen Bedingungen gesehen. Um die verschiedenen Schritte des apoptotischen Spiel zu verstehen und &quot;professionelle&quot; Phagozyten vergleichen - Makrophagen und dendritischen Zellen zu &quot;nicht-professionellen&quot; - Gewebe-resident benachbarten Zellen, in vivo Echtzeit-Bildgebung des Prozesses ist äußerst wertvoll. Hier beschreiben wir ein Protokoll für das Studium apoptotischen Spiel in Live-Drosophila Embryonen. Um die Dynamik der verschiedenen Schritte der Phagozytose verwenden wir spezifische Marker für apoptotische Zellen und Fresszellen folgen. Darüber hinaus können wir überwachen zwei phagocyte Systeme parallel: &quot;professionelle&quot; Makrophagen und &quot;semi-Professional &#39;Glia in der Entwicklung des zentralen Nervensystems (ZNS). Die hier beschriebene Methode nutzt die Drosophila-Embryo als hervorragendes Modell für Echtzeit Untersuchungen des apoptotischen Clearance.

Repräsentative Bilder von einem Film, in dem apoptotischen Zellen mit Annexin V markiert sind, und Glia, Ektoderm und Makrophagen sind mit Simu-cytGFP beschriftet sind in Abbildung 2 dargestellt. Jeder Rahmen ist eine Projektion der 3 Scheiben 2 um jeweils. Der Embryo der Stufe 15 richtig positioniert ist, die die embryonale CNS in der Mitte mit gut markierten Gliazellen (g). Makrophagen (m), die meist außerhalb des ZNS, zeigen starke zytoplasmatische GFP-Expressi...

Watch this Scientific Journal Video about Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis at JoVE.com

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Hier beschreiben wir eine effektive Methode zur Untersuchung der Dynamik apoptotischen Clearance<em&gt; In vivo</em&gt;. Dieses Verfahren verwendet leben<em&gt; Drosophila</em&gt; Embryonen als leistungsfähiges Modell für die Überwachung der Phagozytose von apoptotischen Zellen mit spezifischen Markierung von apoptotischen Zellen und Fresszellen.

Live-Imaging von apoptotischen Ausverkauf während<em&gt; Drosophila</em&gt; Embryogenese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

live-imaging-apoptotic-cell-clearance-during-drosophila

Research

JoVE Journal

Biology

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

13.4K Aufrufe.  Technion-Israel Institute of Technology. Hier beschreiben wir eine effektive Methode zur Untersuchung der Dynamik apoptotischen Clearance In vivo. Dieses Verfahren verwendet leben Drosophila Embryonen als leistungsfähiges Modell für die Überwachung der Phagozytose von apoptotischen Zellen mit spezifischen Markierung von apoptotischen Zellen und Fresszellen.

Serie: Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Live-Imaging von Gliazellen Migration in der Drosophila Eye Imaginalscheibe

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Forschung

Biologie

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Live-Bildgebung Drosophila melanogaster Embryonale Hemocyte Migrationen

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Erstellung von Entwicklungs-und Adult Drosophila Brains und retinae für Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Ein Reverse Genetic Ansatz zur funktionellen Redundanz während der Embryogenese-Test

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Der Ausarbeitung der einzelnen Drosophila Ei Chambers für Live-Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

Live-Imaging von GFP-markierten Proteine ​​in<em&gt; Drosophila</em&gt; Oozyten

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D-Bildgebung von Protein Aggregation in lebenden Zellen

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Die Tomate / GFP-FLP / FRT Verfahren zur Live-Imaging von Mosaic Erwachsene<em&gt; Drosophila</em&gt; Sehzellen

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Die zytologische Analyse der Spermatogenese: Live-und Fest Zubereitungen aus<em&gt; Drosophila</em&gt; Hoden

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

In - vivo - Biosensor - Spuren Non-apoptotischen Caspase - Aktivität in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...